Rapid Phenotyping and Visual Screens Enabled by microfluidics

描述(由申请人提供)：许多神经发育和再生的机制目前尚不清楚；对线虫等模式生物的研究可能会阐明这些机制。线虫是一个众所周知的、常用的模式系统，因为它的基因组测序，简单的神经系统，以及许多突变体和分子试剂的可用性。因此，了解神经元如何极化线虫中的轴突并将其引导到目标位置，可以揭示高等生物体中这些复杂的过程。然而，使用现有技术研究这些机制是困难的。对动物进行表型筛选通常是一个手工过程，而具有微妙表型表达的蠕虫进一步混淆了这个问题，阻碍了线虫研究的进展。线虫研究的长期目标是发现基因和神经活动最终是如何影响行为的。虽然传统遗传学对这一领域做出了巨大贡献，但它的吞吐量有限，而且容易受到人类偏见的影响。这项拟议的工作将通过设计一种微流控系统来推进当前的研究，该系统用于高通量和自动化的神经元极性和轴突引导突变体的表型筛选。该设备将增加基因筛查的吞吐量，并将通过使用表型分析软件来减少人类偏见。为了实现这一目标，我们将改进微流控设备中蠕虫的成像和分类方法。同时，我们将开发能够量化特定神经元特征的图像分析软件。为了验证所提出的技术，我们将使用荧光标记筛选新的突变体。这项拟议的工作意义重大，因为它可以极大地提高目前筛选具有微妙荧光表型的小动物的能力。该项目还将通过发现新的突变体和研究新的途径来推进神经发育研究。拟议的项目不仅对整个科学界有利，而且对年轻研究人员的培训和发展也有好处。由于能够分析大量的线虫基因类型，这项技术与许多领域相关。总体而言，了解任何生物体行为背后的机制将有助于进一步解释人类大脑的奥秘，并可能有助于发现各种疾病的治疗方法。

DESCRIPTION (provided by applicant): The mechanisms of many neural development and regeneration are currently unclear; studies in model organisms such as C. elegans may elucidate these mechanisms. C. elegans are a well known, commonly used model system because of its sequenced genome, simple nervous system, and the availability of many mutants and molecular reagents. Understanding how neurons polarize and guide axons to their targets in C. elegans can therefore shed light on these complex processes in higher organisms. Studying these mechanisms using current technologies, however, is difficult. Performing phenotypic screens of animals is generally a manual process, and worms with subtle phenotypic expression further confound the issue, miting advancement of C. elegans research. The long-term goal in C. elegans research is to discover how genes and neural activity ultimately affect behavior. While traditional genetics has made large contributions to this field, it is limited in throughput and subject to human bias. The proposed work will advance current research by designing a microfluidic system for high-throughput and automated phenotypic screens of neuronal polarity and axon guidance mutants. The device will increase throughput of genetic screens and will reduce human bias by using software for phenotypic analysis. To achieve this goal, we will improve methods for imaging and sorting worms in microfluidic devices. In parallel, we will develop image analysis software capable of quantifying characteristics of specific neuronal features. To validate the proposed technology, we will screen for novel mutants using fluorescent markers. The proposed work is significant because it can dramatically increase current capabilities of screening small animals with subtle fluorescent phenotypes. The project will also advance neural development research by discovering novel mutants and investigating new pathways. The proposed project will not only be beneficial to the scientific community in general, but also to the training and development of a young researcher. With the ability to analyze numerous C. elegans genotypes, this technology is relevant to many fields. In general, understanding the mechanisms behind behavior in any organism will help to further explain the mysteries of the human brain and potentially aid in the discovery of therapeutic treatments for various diseases.